The screening for anticoagulant rodenticide gene VKORC1 polymorphism in the rat Rattus norvegicus, Rattus tanezumi and Rattus losea in Hong Kong

Anticoagulants are a major component of rodenticides used worldwide, which function by effectively blocking the vitamin K cycle in rodents. The rat Vitamin K epoxide Reductase Complex (VKORC) subunit 1 is the enzyme responsible for recycling vitamin K, and five substitution mutations (Tyr139Cys, Tyr139Ser, Tyr139Phe and Leu128Gln and Leu120Gln) located in the VKORC1 could result in resistance to anticoagulant rodenticides. This study carried out a VKORC1-based survey to estimate the anticoagulant rodenticide resistance in three Rattus species (R. losea, R. norvegicus, and R. tanezumi) collected in Hong Kong. A total of 202 rats captured in Hong Kong between 2017 and 2021 were analysed. Sequencing of molecular marker cytochrome c oxidase subunit 1 (COX1) was carried out to assist the species identification, and the identities of 52 lesser ricefield rats (R. losea), 81 common rats (R. norvegicus) and 69 house rats (R. tanezumi) were confirmed. Three VKORC1 exons were amplified from individuals by PCR followed by Sanger sequencing. A total of 47 R. tanezumi (68.1%) contained Tyr139Cys mutation in VKORC1 gene, and half of them were homozygous. None of the collected R. losea and R. norvegicus were detected with the five known substitutions leading to anticoagulant rodenticides resistance, and previously undescribed missense mutations were revealed in each species. Whole genome sequencing was further carried out on some individuals, and single nucleotide polymorphisms (SNPs) were also identified in the introns. This is the first study investigating the situation of anticoagulant rodenticide resistance in the rats collected in Hong Kong. Given that the efficacy of rodenticides is crucial for effective rodent management, regular genetic testing as well as population genomic analyses will be required to both monitor the situation and understand the adaption of different rat haplotypes for integrated pest management. Susceptibility tests for individual rodenticides should also be conducted regularly to assess their effectiveness on local species.

www.nature.com/scientificreports/ Studying mutations of the exonic nucleotide composition or single nucleotide polymorphisms (SNPs) of VKORC1 gene provides crucial information on resistance to As rodenticides and efficacy of pest control [17][18][19] . For instance, ~ 70% of sampled common or Norwegian rats (R. norvegicus) in the United Kingdom carried one of the five known missense mutations (Tyr139Cys, Tyr139Ser, Tyr139Phe and Leu128Gln and Leu120Gln) 20 , while these mutations could confer certain level of resistance to both first and second generation of anticoagulant rodenticides 16,17,19 . In a recent VKORC1-based SNP survey in mice and rats in the United States, it has also been suggested that resistances detected in the 1980s were likely due to mutations of Leu128Ser and Tyr139Cys in house mice (M. musculus domesticus), Arg35Pro in common or Norwegian rats (R. norvegicus), and Tyr25Phe in roof rats (R. rattus) 21 . Nevertheless, limited information was obtained from Asia, including Hong Kong. We therefore collected rodents from Hong Kong and carried out a VKORC1-based survey to estimate the anticoagulant rodenticide resistance situation that could compromise pest management.

Materials and methods
Sampling and DNA extraction. A total of 202 tail samples from dead rodents were provided to The Chinese University of Hong Kong by the Food and Environmental Hygiene Department, The Government of the Hong Kong Special Administrative Region and the City University of Hong Kong. The rodents were captured using traps from different locations in Hong Kong between 2017 and 2021. Tail samples were stored at − 20 °C before further experimental procedures. Genomic DNA extraction was carried out using QIAamp DNA mini kit (QIAgen, Germany) following the manufacturer's instructions. In brief, 0.02 g of tail tissue were homogenized and incubated with proteinase K at 55 °C for 2 h. The quantity and quality of DNA were determined by Nanodrop (Ratio of 260/280 ~ 1.8 and 260/230 ~ 1.8-2.0) and gel electrophoresis under Gel Doc™ EZ imager (Bio-Rad), respectively. Species identification. Molecular identification was carried out via the polymerase chain reaction (PCR) of mitochondrial DNA cytochrome c oxidase subunit 1 (COX1) gene using a model of T100™ thermocycler (Bio-Rad). COX1 gene was amplified using rodent specific primer BatL5310 (5′-CCT ACT CRG CCA TTT TAC CTA TG-3′) and R6036R (5′-ACT TCT GGG TGT CCA AAG AAT CA-3′) 22  The amplified products (762 bp) were confirmed on 1% agarose gel stained as well as Sanger sequencing (BGI Genomics Company Hong Kong). Obtained sequences were edited with software SnapGene Viewer, and aligned using MEGA X for phylogenetic analysis (Neighbour-joining method and 1000 bootstrap replications). VKORC1 sequence analysis. All three exons of VKORC1 gene were amplified following a previous study using specific primers: (Exon1 forward: 5′-GTG GCG GGT TCT TCC CTC-3′; Exon 1 reverse: 5′-GAC TCC AAA ATC ATC TGG CAA CC-3′), (Exon 2 forward: 5′-AAG AGT AGG GGAC AAG GTG GC-3′; Exon 2 reverse: 5′-GGG TCA CCA AGA CAT GAG GTG-3′) and (Exon 3 forward: 5′-TTT CAC CAG AAG CAC CTG CTG CC-3′; Exon 3 reverse: 5′-ACA CTT GGG CAA GGC TCA TGT G-3′) 13 . The amplified products were confirmed on 2% agarose gel stained as well as Sanger sequencing (BGI Genomics Company Hong Kong). SNP of each exon sequence was compared to the available sequence from NCBI database (VKORC1 GenBank accession no. AY423047) with MEGA X software. BlastX searches with adjusted sequences were also carried out to locate any missense mutation. Homozygous and heterozygous genotypes of five published missense mutations on exon 3 were further confirmed on each chromatogram using SnapGene Viewer.
Genome sequencing of selected individuals. DNA of R. norvegicus and R. tanezumi from 4 localities including Yuen Long (YL_2, YL_3), Wan Chai (Wch_1, Wch_2), Kwun Tong (KTo_4, KTo_5) and Islands (Is_1, Is_6) were proceeded with low-coverage whole genome sequencing (Table 1). Raw sequenced reads were mapped to the R. norvegicus reference genome (GenBank assembly accession: GCF_000001895.5) and SNPs were called with Genome Analysis Toolkit (GATK) 23 . The SNP dataset was annotated with the gene models of  1). Greater genetic diversity was also observed in the COX1 of R. norvegicus than the two other captured species. Among the five previous reported mutations in VKORC1 exon 3 reported elsewhere in the world (Tyr139Cys, Tyr139Ser, Tyr139Phe and Leu128Gln and Leu120Gln), only Tyr139Cys mutation was found in the R. tanezumi samples but not in the other collected species.
In the 69 collected R. tanezumi, 47 of them (68.1%) were found to carry Tyr139Cys mutations with 25 homozygotes and 22 heterozygotes. Details of their sampling locations and number of mutations are summarised in Table 2    In addition to the known Y139C mutation, nonsynonymous mutations were also found from one R. norvegicus sample and one R. losea sample, respectively. Further, six synonymous mutations were also found among three species. Details are provided in Supplementary information S1.
Besides the exons, a total of nine SNPs was revealed locating at the introns, with three coming from R. norvegicus and the other six from R. tanezumi (Table 4).

Ethics declaration. Animal ethics approval was granted by the Animal Research Ethics Sub-Committee of
City University of Hong Kong. All methods were carried out in accordance with relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines.   www.nature.com/scientificreports/ from two locations only. Despite R. rattus and R. tanezumi were well known to be difficult to be morphologically differentiated from one another 22,25 , given the previous and present studies were carried out at different time (more than ten years) and places using different collection method, it is unclear whether the situation represents misidentification, distribution in different biotopes, different collection methods, or changes in dominant rodent species spatiotemporally.
In the limited studies carried out on anticoagulant rodenticide resistance in Asia, a relatively low warfarinresistance rate (11%, 4 out of 36 samples) was determined by lethal feeding test in R. tanezumi collected from mainland China ten years ago 26 . It should be noticed that the use of anticoagulant rodenticides in China was believed to have started in the early 1980s 27 , which has a shorter history than other places in the world. A recent study also suggested a low anticoagulant rodenticide resistance rate in R. norvegicus collected from two cities in mainland China 28 . This study, based on the VKORC1 gene survey, discovered 68.1% of R. tanezumi in Hong Kong carried the Tyr139Cys mutation.
Previous studies suggested that the Tyr139Cys mutation could confer resistance to first-and second-generation anticoagulant rodenticides including bromadiolone and difenacoum in Norway rat and house mouse 19 . Given the relationships between anticoagulant rodenticide resistance and the Tyr139Cys mutation in R. tanezumi has not been tested, the cause and significance of such mutation being only observed in R. tanezumi but not in R. losea and R. norvegicus remains to be revealed. In case if the Tyr139Cys mutation in R. tanezumi also confer certain type of anticoagulant rodenticide resistance, other substances such as difethialone and flocoumafen could to be used 29 . Regarding to the rodent nuisance in Hong Kong, anticoagulant compound is more desirable and safer rodenticide for controlling rodents compared with acute poison within the densely populated urban area. Anticoagulant compound has been widely adopted by both private and public pest control operators. Currently, there is no statutory regulation to monitor the use of rodenticide from local pest control operators, however, the low efficacy of certain compound and good prevention practice should be aware in order to decrease the influence of rodent problems.
This study also revealed other SNP variants not documented previously, for instances, two synonymous SNPs and one nsSNP (Trp101Cys) in R. losea. It is also worth noting that no SNPs located in exon 1 identified from the nine R. losea samples were Arg58Gly mutation which confer anticoagulant rodenticide resistance 30 . These data bring up the issues that there are huge gaps in knowledge regarding the origin, introduction, genetic diversity, population connectivity of Rattus between different places in Asia, as well as the relationships of mutations brought in to VKORC1 genes and their anticoagulant rodenticide resistance of different Rattus population from different places in Asia.

Conclusion
This study provided the baseline information of rodenticide resistance status and distribution of 202 rodents belonging to 3 Rattus species in Hong Kong. The investigation indicates a distinctive anticoagulant rodenticide resistance pattern. The relatively high Tyr139Cys mutation found in VKORC1 gene of R. tanezumi suggested further susceptibility tests will be needed to reveal whether they are resistance to individual anticoagulant rodenticide and to ensure effectiveness on local species. Regular genetic testing and genomic analyses will also be required to understand the situations of rodent populations for integrated pest management.

Data availability
The raw reads generated in this study have been deposited to the NCBI database under the BioProject accession PRJNA723168.